annualreviewofcellanddevelopmentalbiology … · 2019. 7. 15. · cb35ch12_gu arjats.cls july4,2019...

CB35CH12_Gu ARjats.cls July 4, 2019 13:53

Annual Review of Cell and Developmental Biology

Development and Cell Biologyof the Blood-Brain BarrierUrs H. Langen,1,∗ Swathi Ayloo,1,∗ and Chenghua Gu1

Department of Neurobiology, Harvard Medical School, Boston, Massachusetts 02115, USA;email: [email protected]

Annu. Rev. Cell Dev. Biol. 2019. 35:12.1–12.23

The Annual Review of Cell and Developmental Biologyis online at cellbio.annualreviews.org

https://doi.org/10.1146/annurev-cellbio-100617-062608

Copyright © 2019 by Annual Reviews.All rights reserved

∗These authors contributed equally

Keywords

blood-brain barrier, neurovascular unit, central nervous system, endothelialcells, pericytes, astrocytes

Abstract

The vertebrate vasculature displays high organotypic specialization, withthe structure and function of blood vessels catering to the specific needsof each tissue. A unique feature of the central nervous system (CNS)vasculature is the blood-brain barrier (BBB). The BBB regulates substanceinflux and efflux to maintain a homeostatic environment for proper brainfunction. Here, we review the development and cell biology of the BBB,focusing on the cellular and molecular regulation of barrier formationand the maintenance of the BBB through adulthood. We summarizeunique features of CNS endothelial cells and highlight recent progressin and general principles of barrier regulation. Finally, we illustrate whya mechanistic understanding of the development and maintenance of theBBB could provide novel therapeutic opportunities for CNS drug delivery.

12.1Review in Advance first posted on July 12, 2019. (Changes may still occur before final publication.)

Ann

u. R

ev. C

ell D

ev. B

iol.

2019

.35.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

g A

cces

s pr

ovid

ed b

y H

arva

rd U

nive

rsity

on

07/1

5/19

. For

per

sona

l use

onl

y.

mailto:[email protected]

https://doi.org/10.1146/annurev-cellbio-100617-062608


Contents

1. INTRODUCTION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.22. UNIQUE BARRIER PROPERTIES OF CNS ENDOTHELIAL CELLS . . . . . . 12.3

2.1. Specialized Tight Junctions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.32.2. Transporters at the Blood-Brain Barrier . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.42.3. Low Rates of Transcytosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.52.4. Limited Immune Cell Trafficking at the Blood-Brain Barrier . . . . . . . . . . . . . . . 12.7

3. BARRIER REGULATION BY THE NEUROVASCULAR UNIT . . . . . . . . . . . . . 12.73.1. Pericytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.73.2. Astrocytes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.93.3. Basement Membrane of the Neurovascular Unit . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.10

4. DEVELOPMENT OF THE BLOOD-BRAIN BARRIER . . . . . . . . . . . . . . . . . . . . . 12.114.1. Barriergenesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.114.2. Wnt Signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.13

5. OUTLOOK . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12.15

1. INTRODUCTION

The blood-brain barrier (BBB) is the interface that separates neural tissue from circulating blood.The BBB comprises a single layer of endothelial cells that forms the blood vessel wall and main-tains a safe and homeostatic milieu for proper neuronal function and synaptic transmission. Im-paired barrier function is associated with neurodegenerative diseases, including multiple sclerosis,Alzheimer’s disease, and Parkinson’s disease (Sweeney et al. 2018,Zhao et al. 2015).Conversely, theBBB is a major obstacle for drug delivery into the CNS, impeding the treatment of neurologicaldiseases, including neurodegenerative diseases, psychiatric disorders, brain infections, and braintumors (Banks 2016, Pardridge 2012). Despite the importance of the BBB, our understandingof the fundamental mechanisms underlying the formation and maintenance of the BBB remainslimited.

Our limited understanding of the BBB can be attributed to its complexity. The BBB is local-ized to CNS endothelial cells that build the vessel wall and display unique cell biological features.These features distinguish these cells from peripheral endothelial cells. First, CNS endothelialcells have specialized tight junctions (TJs) to prevent free paracellular passage through the vesselwall. Second, they express designated transporters to regulate the dynamic influx and efflux of spe-cific substrates. Third, they display extremely low rates of transcellular vesicle trafficking, termedtranscytosis, to limit transcellular transport through the vessel wall. Lastly, CNS endothelial cellshave low expression levels of leukocyte adhesion molecules (LAMs) to limit the entry of immunecells into the brain. Recent studies have also implicated the glycocalyx, a negatively charged denselayer of carbohydrates on the luminal side, in preventing immune cell entry, acting as the first lineof defense in protecting the brain (Kolarova et al. 2014, Kutuzov et al. 2018) (Figure 1).

However, barrier properties are not intrinsic to endothelial cells. BBB formation and main-tenance depend on local perivascular cells and the neuronal environment of the CNS (Armuliket al. 2010, Daneman et al. 2010, Janzer & Raff 1987, Stewart &Wiley 1981). Blood vessels in thebrain are surrounded by pericytes and astrocytes, which serve as the interface between endothelialcells and neurons. Together, these cells form the neurovascular unit (NVU) (Iadecola 2004, 2017;Zhao et al. 2015) (Figure 1).

12.2 Langen • Ayloo • GuReview in Advance first posted on July 12, 2019. (Changes may still occur before final publication.)

Ann

u. R

ev. C

ell D

ev. B

iol.

2019

.35.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

g A

cces

s pr

ovid

ed b

y H

arva

rd U

nive

rsity

on

07/1

5/19

. For

per

sona

l use

onl

y.


a Neurovascular unit

Lumen

EC

PCBM

AE

BM

EC

Leukocyte

b Barrier properties of brain ECs

Brainparenchyma

Specializedjunctions

Lowvesiculartransport

Distinctglycocalyx;low LAMexpression

Specific influxand effluxtransporters

1

2

3

4

Lumen

Figure 1

The neurovascular unit. (a) Schematic illustration of a cross section through the neurovascular unit.Capillary endothelial cells (EC) forming the vessel walls are tightly wrapped by pericytes (PC). These cellsare embedded in the basement membrane (BM) and are ensheathed by astrocytic endfeet (AE). (b) Schematicof the CNS endothelium highlighting the four unique properties of the blood-brain barrier. LAM denotesleukocyte adhesion molecule.

In this review,we provide an overview of the cellular andmolecular features of the BBB.Wefirstelucidate the specialization of CNS endothelial cells and discuss the cell biology underlying theirunique characteristics.We then illustrate the role of other cell types in theNVU that contribute tobarrier function.Next, we lay out the development of the BBB, focusing on the signaling pathwaysand mechanisms that establish the barrier. Finally, we discuss critical questions in the field thatrequire further investigation.

2. UNIQUE BARRIER PROPERTIES OF CNS ENDOTHELIAL CELLS

2.1. Specialized Tight Junctions

Initial tracer injection experiments revealed that brain endothelial cells have specialized TJs thatprevent the intercellular passage of tracers from the circulation into the brain parenchyma (Reese& Karnovsky 1967). Using ultrastructural electron microscopy analysis, this study revealed thatthe entry of the injected tracer is sharply halted at TJs between two neighboring endothelial cells,highlighting two aspects: (a) CNS endothelial cells are the site of the BBB, and (b) specialized TJsare important for BBB integrity. Subsequent studies revealed that CNS TJs are specialized to sealcontact sites between endothelial cells to prevent paracellular transport of molecules into the CNSand that adherens junctions promote cell-cell adhesion (Brightman & Reese 1969, Lampugnaniet al. 1992). Earlier studies focusing on junctions in epithelial cells informed much of our under-standing about TJs in general and formed the foundation for our understanding of TJs in CNSendothelial cells. Epithelial cells lining peripheral tissues have three kinds of junctions: TJs gener-ally found on the apical side of epithelial cells, desmosomes on the basolateral side, and adherensjunctions found in between desmosomes and adherens junctions (Farquhar & Palade 1963). Incontrast, CNS endothelium lacks desmosomes and has a different spatial distribution of junctions

www.annualreviews.org • Blood-Brain Barrier 12.3Review in Advance first posted on July 12, 2019. (Changes may still occur before final publication.)

Ann

u. R

ev. C

ell D

ev. B

iol.

2019

.35.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

g A

cces

s pr

ovid

ed b

y H

arva

rd U

nive

rsity

on

07/1

5/19

. For

per

sona

l use

onl

y.


than do epithelial cells. Specifically, TJs are embedded within adherens junctions and are foundthroughout the cleft between endothelial cells (Schulze & Firth 1993).

The four classes of TJ proteins in the CNS are occludin, tricellulins, claudins, and junctionaladhesion molecules ( JAMs).Occludin (encoded byOcln) is enriched in CNS endothelium and wasthe first transmembrane protein shown to localize exclusively to TJs (Furuse et al. 1993). How-ever,Ocln−/− mice develop normal TJs, despite having other abnormalities like calcification of thebrain (Saitou et al. 1998, 2000). A closer examination of Ocln−/− mice revealed mislocalization oftricellulin proteins from tricellular TJs (where three cells meet) to bicellular TJs (between twocells) (Ikenouchi et al. 2008). These findings suggest that the role of Occludin in TJ formation iscomplex, and given the structural similarities between tricellulins and Occludin (Ikenouchi et al.2005), tricellulins may compensate for Occludin deficiency, providing an explanation for the ap-parently normal TJs in Ocln−/− mice. Consistent with this idea, recent work has demonstrateda role for Lsr, a tricellulin protein enriched at the BBB, in the formation of a functional barrier(Sohet et al. 2015).

The surprising finding of functional barriers in Ocln−/− mice fueled the continued search forother TJ proteins, leading to the discovery of claudins (Furuse et al. 1998). Of the 27 mammalianclaudin genes, the gene encoding Claudin-5 (Cldn5) is highly enriched in CNS endothelial cells(Morita et al. 1999). Cldn5−/− mice exhibit a size-selective increase in barrier permeability (Nittaet al. 2003), implicating Claudin-5 as a key component of BBB regulation. These mice displaynormal vascular development and show no gross abnormalities in peripheral tissues but die shortlyafter birth, likely due to an impaired BBB (Nitta et al. 2003). Finally, JAMs are transmembraneTJ proteins belonging to the immunoglobulin superfamily that regulate leukocyte transmigrationacross cellular barriers (Martin-Padura et al. 1998).

While several TJ proteins are decreased in pathological conditions, our understanding of thebasic biology of these proteins in the context of the BBB is still rudimentary. Intracellular traf-ficking patterns of these proteins and their turnover rates in the CNS endothelium are unknown.Generating CNS endothelium–specific conditional knockout mouse models for these proteins todetermine their contribution to BBB formation and maintenance will provide insight into thespecific roles of such proteins in barrier function.

2.2. Transporters at the Blood-Brain Barrier

The highly selective nature of the BBB and the high metabolic demand of the brain necessitateother routes of entry for various nutrients to feed and nurture the brain. Metabolic supply isachieved via several influx and efflux transporters that are expressed on the surface of CNSendothelial cells and that drive the active transport of specific solutes and metabolites into thebrain. Transport across the BBB can generally be classified into four categories: (a) passive dif-fusion of lipid-soluble molecules such as oxygen, (b) carrier-mediated transport via solute carriertransporters (SLCs), (c) selective transport via ATP-binding cassette transporters (ABCs), and(d) vesicular trafficking by transcytosis (Section 2.3). Here we briefly discuss transporters ex-pressed at the BBB. Many of these transporters are ATPases and ATP-binding pumps, indicatinga high energy demand to maintain the barrier. Consistent with this, CNS endothelial cells havefour- to fivefold more mitochondria relative to peripheral endothelial cells (Oldendorf et al. 1977).

SLCs are a large class of transporters that facilitate the uptake of nutrients, including glucose,amino acids, ions, and fatty acids (Campos-Bedolla et al. 2014). Owing to the structural complex-ity and the genetic redundancy of the more than 400 genes in the SLC family, SLCs have beenrelatively difficult to study. One of the best-studied SLCs is SLC2A1, or Glut1 (glucose trans-porter 1), which transports glucose. The BBB is highly enriched in Glut1, and patients with Glut1


Ann

u. R

ev. C

ell D

ev. B

iol.

2019

.35.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

g A

cces

s pr

ovid

ed b

y H

arva

rd U

nive

rsity

on

07/1

5/19

. For

per

sona

l use

onl

y.


deficiency present with seizures, microcephaly, developmental delays, and ataxia (De Vivo et al.1991). While Slc2a1−/− mice die at embryonic stages, mice haploinsufficient for Slc2a1 recapitu-late the features seen in human patients (Wang et al. 2006). Recent work has also shown that lackof Glut1 causes barrier breakdown and prevents clearance of amyloid plaques, thus contributingto the progression of Alzheimer’s disease (Winkler et al. 2015).

SLC7A5, or LAT1 (large neutral amino acid transporter 1), is another well-studied transporterin this family. LAT1 transports branched amino acids into the brain and, like Glut1, is highlyenriched in CNS endothelial cells. Recent identification of mutations in LAT1 associated withautism spectrum disorder gave further insight into the role of LAT1. Mice lacking endothelialLAT1 have developmental and neurological abnormalities (Tarlungeanu et al. 2016). These micealso display altered amino acid levels in the brain, resulting in reduced mRNA translation withslower protein synthesis. Besides amino acid uptake transporters, the BBB also expresses SLCsthat drive the net efflux of amino acids like glutamate and aspartate, which are excitotoxic athigh levels. These SLCs are excitatory acidic amino acid transporters (EAATs) that are expressedon the abluminal side of CNS endothelial cells (O’Kane et al. 1999) and that export gluta-mate from the brain parenchyma to maintain low levels of glutamate in the brain extracellularfluid. Thus, the roles of these transporters highlight the importance of regulating amino acidconcentrations within the brain for neuronal homeostasis.

In contrast to most nutrient transporters, ABC efflux pumps actively clear out metabolic wastefrom the CNS and hence are usually considered molecular defense systems that protect the brainfrom exogenous and toxic substances. Among these transporters, P-glycoprotein (P-gp) has beeninvestigated extensively owing to its role in amyloid β (Aβ) efflux from the brain parenchyma.Aβ are small peptides that can form plaques in the brain if they are not cleared, a phenomenonseen widely in Alzheimer’s patients. Initial studies observed that P-gp is on the luminal side ofBBB capillaries (Beaulieu et al. 1997, Cordon-Cardo et al. 1989) and promotes the secretion ofAβ (Lam et al. 2001). Mice lacking P-gp not only have increased deposition of Aβ (Cirrito et al.2005) but also display increased sensitivity to several drugs (Schinkel et al. 1996), highlightingthe role of P-gp in preventing drugs from entering the brain. Consistently, multiple studies havereported decreased levels of P-gp and other ABC efflux pumps in postmortem brain tissue samplesof Alzheimer’s disease patients ( Jeynes & Provias 2011, Wijesuriya et al. 2010), implicating thedecreased levels of those pumps in promoting disease progression.

Overall, our understanding of the transporters at the BBB is still in the early stages. SLCsare thought to make up approximately 10% of the human genome (Hediger et al. 2013), yet theprecise roles of most of these proteins are not known. In the case of efflux pumps, it is unclearwhat triggers their initial loss contributing to disease progression. General questions that remainin this field include: What portion of the nutrients provided by the transporters is consumed byendothelial cells themselves versus other cell types in the NVU? Furthermore, what is the fate ofthese nutrients once taken up by endothelial cells, and how are such nutrients transported to othercell types in the brain? These aspects are completely unknown for most transporters at the BBB.

2.3. Low Rates of Transcytosis

Besides having designated transporters to deliver specific nutrients to the brain, CNS endothelialcells also have very low rates of vesicle-mediated transcellular trafficking, a process termed tran-scytosis (Reese & Karnovsky 1967). Recent work demonstrated that these low rates result fromthe active inhibition of transcytosis in CNS endothelial cells. The mechanisms regulating this in-hibition at the BBB were largely unknown until recent studies established Mfsd2a as an inhibitorof transcytosis in regulating permeability of the BBB (Andreone et al. 2017, Ben-Zvi et al. 2014,


Ann

u. R

ev. C

ell D

ev. B

iol.

2019

.35.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

g A

cces

s pr

ovid

ed b

y H

arva

rd U

nive

rsity

on

07/1

5/19

. For

per

sona

l use

onl

y.


Chow & Gu 2017). Mfsd2a is a lipid transporter (Nguyen et al. 2014) enriched in CNS endothe-lial cells, andMfsd2a−/− mice have increased caveolae-mediated vesicular trafficking, causing leakybarriers (Andreone et al. 2017, Ben-Zvi et al. 2014, Chow & Gu 2017). Andreone et al. (2017) in-vestigated the specific mechanism by which Mfsd2a inhibits transcytosis: Intriguingly,Mfsd2a, byits lipid transport function, establishes a unique lipid composition in the CNS endothelial cellplasma membrane that inhibits caveolae vesicle formation, thereby suppressing transcytosis.

Although CNS endothelial cells have limited transcytosis, some macromolecules, such as in-sulin, albumin, and iron-bound transferrin, lack specific transporters at the BBB and thus en-ter the brain by transcytosis. The passage of these macromolecules demonstrates how the BBBcan precisely regulate the transcytotic pathway to allow for the transendothelial passage of spe-cific molecules via either receptor-mediated transcytosis or adsorptive transcytosis. In the for-mer, receptors on the luminal cell surface bind to cargo or their ligand. This complex is thenendocytosed and trafficked within the endothelial cell. Adsorptive transcytosis, in contrast, isreceptor-independent vesicular trafficking and depends on charged interactions between the lig-and and the glycocalyx of endothelial cells. In both cases, after the first step of vesicle formation,the vesicles are trafficked through endosomes and exocytosed into the brain parenchyma. Whileclathrin-mediated transcytosis and caveolae-mediated transcytosis are the two major endocyticpathways, other kinds of vesicular structures have been reported at the ultrastructural level byusing electron microscopy analyses (Hansen &Nichols 2009). Furthermore, various adaptor pro-teins play essential roles in each step of these processes. However, it is unclear what determineswhether a molecule will be transcytosed and delivered into the brain or sorted into lysosomes fordegradation.

On the therapeutic front, researchers have sought to harness receptor-mediated transcytoticpathways, particularly the transferrin receptor (TfR)-mediated transcytosis pathway, to facilitatedrug delivery into the brain (Yu & Watts 2013). Tfr-mediated transcytosis has been an attrac-tive system to explore the possibilities of facilitating CNS drug delivery. Antibodies targetingTfR preferentially bind to brain capillaries ( Jefferies et al. 1984). After binding, the antibody-TfRcomplex is shuttled into the brain parenchyma via transcytosis (Friden et al. 1991). Bispecific an-tibodies targeting TfR and Aβ have been employed to promote the delivery of these antibodiesinto the CNS and to promote the clearance of Aβ plaques (Bien-Ly et al. 2014, Niewoehneret al. 2014). These studies revealed that both the affinity and the valency of antibody bind-ing to the receptor are important in determining the efficient delivery of the antibody into theCNS.

Recent studies have also demonstrated that upregulation of transcytosis is an initial step in cer-tain models of brain disorders. Studies investigating the BBB in stroke and brain injury modelshave revealed that a few hours post–brain insult, transcytotic vesicles in endothelial cells are up-regulated, causing barrier breakdown (Haley & Lawrence 2017, Knowland et al. 2014, Sadeghianet al. 2018). Consistent among all these studies are also the observations that leakage via transcel-lular pathways precedes breakdown of TJs.While these results may apply only to specific modelssuch as stroke, it is worth examining what factors cause the early increase in transcytosis at theBBB. Identification of these factors will allow for the development of strategies to prevent barrierbreakdown.

On the CNS drug delivery front, the discovery of BBB regulators like Mfsd2a provides anentry point for the development of compounds that specifically target proteins to open the bar-rier for CNS drug delivery. For example, inhibiting Mfsd2a to upregulate transcytosis at the BBBcould provide a platform for drug delivery into the brain. Thus, identification of molecules uniqueto CNS barriers will provide a parts list to effectively harness transcytosis pathways for drugdelivery.


Ann

u. R

ev. C

ell D

ev. B

iol.

2019

.35.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

g A

cces

s pr

ovid

ed b

y H

arva

rd U

nive

rsity

on

07/1

5/19

. For

per

sona

l use

onl

y.


2.4. Limited Immune Cell Trafficking at the Blood-Brain Barrier

The CNS has low levels of immune cell surveillance relative to peripheral tissues. Initial studiesrevealed that extremely low numbers of leukocytes, particularly T cells, enter the brain (Hauseret al. 1983,Hickey et al. 1991). Further experiments demonstrated that CNS endothelial cells nor-mally express low levels of LAMs (Rossler et al. 1992) and that LAMs are significantly upregulatedin autoimmune diseases such as multiple sclerosis (Cannella et al. 1990, Raine et al. 1990).

Leukocyte entry into tissues is a highly orchestrated process involving multiple steps and isan early event of pathogenesis in autoimmune diseases. Briefly, leukocytes first survey the vas-cular wall, and signaling molecules such as selectins mediate their initial capture onto the vesselwall. This capture triggers the firm adhesion of the leukocyte to the endothelium via interac-tion of leukocyte integrins with endothelial membrane proteins such as ICAM-1, ICAM-2, andVCAM-1. Following leukocyte adhesion, diapedesis through the vessel wall occurs through eitherthe paracellular or the transcellular route, leading to their entry into the CNS parenchyma. Theentry of leukocytes into the CNS and their subsequent binding to antigen-presenting cells withinthe brain cause the release of cytokines and chemokines. These molecules in turn recruit moreleukocytes, thus acting in a positive feedback manner for further infiltration of these cells intothe CNS. For detailed reviews on immune cell entry into and trafficking at the BBB, please seeEngelhardt & Wolburg (2004) and Ransohoff & Engelhardt (2012).

Recent studies have begun to address which cell types in the brain first sense inflammation sig-nals and how these processes impact the BBB.With the initiation of infection,mural cells of bloodvessels (pericytes and smooth muscle cells) release the CCL2 chemokine, triggering neuroinflam-matory responses acting as sensors of the initial infiltration of immune cells (Duan et al. 2018).Another recent study exploring the effects of stress on neuroinflammation and the BBB revealedthat, in mouse models of social defeat, there is a decrease in Claudin-5 expression in some partsof the brain; this decrease in turn promotes the infiltration of cytokines such as interleukin-6 intothe CNS parenchyma (Menard et al. 2017).

Collectively, these studies highlight the complexity of the neuro-immune axis. We currentlyknow just a fragment of the multistep, highly coordinated process of how initial leukocyte entryinto the CNS causes neuroinflammation and pathology. Further studies are required to delineatethe manifold cell types and multiple signaling components that regulate the neuro-immune axis.

3. BARRIER REGULATION BY THE NEUROVASCULAR UNIT

The characteristics described above reflect unique properties of the BBB in endothelial cells.How-ever, early transplantation experiments have shown that these properties are not intrinsic to theCNS endothelium (Stewart & Wiley 1981). Rather, such properties are induced by the CNS en-vironment. Indeed, while the BBB is localized to CNS endothelial cells, it requires multiple celltypes, such as pericytes and astrocytes, working in concert to form a functional barrier. Together,these cells form the NVU, which induces and regulates barrier formation (Figure 1) and whichwe discuss below.

3.1. Pericytes

Pericytes are mural cells enwrapping capillary blood vessels on their abluminal side. Structurally,pericytes extend processes from their cell body, covering several endothelial cells. In contrastto peripheral tissues, the brain and the retina have the highest pericyte–to–endothelial cell ra-tio (Frank et al. 1987, 1990). Pericytes are embedded within the basement membrane (BM) ofcapillary endothelial cells and are thus centrally positioned between endothelial cells, astrocytes,


Ann

u. R

ev. C

ell D

ev. B

iol.

2019

.35.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

g A

cces

s pr

ovid

ed b

y H

arva

rd U

nive

rsity

on

07/1

5/19

. For

per

sona

l use

onl

y.


and neurons. Although pericytes were first described in the 1870s (Eberth 1871, Rouget 1873),studying them has been difficult due to the lack of specific markers. It is now generally acceptedthat pericytes are NG2 positive, platelet-derived growth factor receptor β (PDGFRβ) positive,and alpha smooth muscle actin negative (Armulik et al. 2011). Recently, the fluorescent Nissl dyeNeuroTrace 500/525 was shown to specifically label pericytes in live mice, allowing for in vivoimaging (Damisah et al. 2017).

The physical apposition of pericytes and endothelial cells facilitates signaling between them.This signaling plays a role in vessel formation, vessel maturation, pericyte recruitment, and peri-cyte coverage (Gaengel et al. 2009). PDGF-B signaling is one such pathway. PDGF-B secreted byendothelial cells recruits pericytes to blood vessels by binding to PDGFRβ, a cell surface proteinon pericytes (Hellstrom et al. 1999, Lindahl et al. 1997).While deletion of either gene (Pdgfb andPdgfrb) is perinatally lethal (Leveen et al. 1994, Soriano 1994), studies using viable mouse modelsdefective in PDGF-B signaling demonstrated a critical role for pericytes in the formation of theBBB (Armulik et al. 2010,Daneman et al. 2010). These mice have decreased pericyte coverage andcontinue to have a dysfunctional barrier through adulthood (Bell et al. 2010). These studies alsorevealed that an intact barrier is highly reliant on the appropriate pericyte coverage of endothe-lial cells. Consistent with this notion, overexpression of PDGF-B causes an increase in pericytenumbers and results in impaired development of the retinal vasculature, resembling proliferativeretinopathy (Edqvist et al. 2012, Seo et al. 2000).

While the role of pericytes in barrier formation is now well acknowledged, their role in themaintenance of the barrier through adulthood remained unknown until a recent study examinedthe role of PDGF-B signaling in adult mice (Park et al. 2017). In contrast to the role of this path-way in recruiting pericytes during development, endothelium-specific deletion of PDGF-B duringadulthood did not cause either (a) loss of pericytes from the endothelium or (b) the breakdownof the blood-retinal barrier (BRB) or the BBB. Park et al. (2017) also induced diphtheria toxinexpression in adult pericytes to specifically cause their ablation and found that pericyte depletionhad no effect on the adult BRB. However, the authors did note that pericyte-ablated adult reti-nas were more susceptible to leakage under stress or injury conditions. These findings suggestthat, although pericytes are required for barrier formation, they may be dispensable for barriermaintenance through adulthood.

All mouse models used in these studies are mutants causing reduction or ablation of an entirecell type (pericytes) in the NVU. Identifying pericyte genes that do not affect pericyte recruitmentto capillaries but play a role in barrier formation will be crucial in dissecting the specific mecha-nisms of how pericytes confer barrier properties to CNS endothelium.One known regulator is thetranscription factor Foxf2, which is specifically expressed in CNS pericytes (Reyahi et al. 2015).Foxf2−/− mice have decreased PDGFRβ signaling, yet they have significantly increased pericytedensity and fail to form a functional BBB. In stark contrast to the study by Park et al. (2017),Reyahiet al. (2015) found that Foxf2 deletion in adult mice resulted in severe BBB leakage, revealing therole of a pericyte-specific gene in BBB maintenance. Additionally, Reyahi et al. (2015) made theintriguing observation that BBB integrity is not affected during the first 3 weeks after Foxf2 dele-tion but is severely compromised after 6 weeks. These findings implicate a role for pericytes inbarrier maintenance as well.

Collectively, these studies reveal the following compelling ideas. First, pathways in addition toPDGFRβ signaling promote pericyte migration and recruitment. Second, BBB breakdown weeksafter Foxf2 deletion suggests a gradual deterioration of the components regulating barrier in-tegrity, likely due to slow turnover rates of these molecules. In light of these findings, the timingof analysis for a functional barrier after temporal gene/pericyte ablation could impact the con-clusion. For example, although Park et al. (2017) did not observe a barrier defect at 3 weeks after


Ann

u. R

ev. C

ell D

ev. B

iol.

2019

.35.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

g A

cces

s pr

ovid

ed b

y H

arva

rd U

nive

rsity

on

07/1

5/19

. For

per

sona

l use

onl

y.


gene/pericyte ablation, the barrier may break down several weeks after pericyte ablation. Third,pericyte loss could make CNS barriers susceptible to breakdown under stress, as shown by Parket al (2017). Together, all these ideas call for a careful investigation into the role of pericytes inbarrier function.

Pericyte loss is also correlated with BBB breakdown in Alzheimer’s disease (Sagare et al.2013). Pericytes play an active role in the clearance of amyloid aggregates via expression of LRP1(Kanekiyo et al. 2012), an Aβ clearance receptor (Deane et al. 2004, Shibata et al. 2000). CNS per-icytes also have a remarkable concentration of acid phosphatase–positive lysosomes (Broadwell &Salcman 1981), which likely indicates robust phagocytic and degradative capabilities. Consistentwith this, studies have demonstrated clearance of cellular debris by pericytes (Castejon et al. 2005,Mazlo et al. 2004). Lastly, several studies have indicated the multipotent differentiation capacityof pericytes (Birbrair et al. 2015, Dore-Duffy 2008). However, we know very little about pericytedifferentiation and turnover rates. The last decade in barrier research has uncovered a significantrole for pericytes in barrier function. It is becoming increasingly clear that we are only beginningto appreciate the role of pericytes in the CNS, and future studies will further evolve our under-standing of pericytes in terms of their physiological functions and their role in pathophysiology.

3.2. Astrocytes

Astrocytes ensheathing capillaries constitute the most abluminal layer of the NVU. These cellscontact the outer BM of the brain vasculature via polarized endfeet that express the water chan-nel aquaporin 4 (Aqp4). Astrocyte endfeet produce extracellular matrix (ECM) proteins that con-tribute to the unique BM of brain capillaries (see Section 3.3). Given the close contact of astrocyteendfeet with brain capillaries, it is intuitive to assume that they play a role in BBB formation ormaintenance.However, it is unlikely that astrocytes contribute to BBB formation in development,because they appear postnatally in the brain, well after the barrier has sealed (Yang et al. 2013).Rather, they are likely important for barrier maintenance.

An early transplantation study suggests that an astrocytic environment is sufficient to inducebarrier properties in newly forming vessels ( Janzer & Raff 1987). In this study, astrocytes weretransplanted into the eyes of rats. Two weeks after transplantation, newly formed vessels in astro-cyte aggregates on the surface of the iris contained a functional barrier. In contrast, transplanta-tion of meningeal cells also led to vascularization of cell aggregates, but these vessels displayed aleaky phenotype ( Janzer & Raff 1987). Yet it is unclear whether the astrocytes themselves inducedbarrier properties in endothelial cells or whether they recruited additional cells that induced thefunctional barrier. Along the same line, blood vessels in the retina develop a functional barrierpostnatally (Chow & Gu 2017), when astrocytes are already present there. The growing vesselsin the retina display a leaky barrier phenotype in the presence of astrocytes (Chow & Gu 2017),indicating that astrocytic signals alone are not sufficient to induce barrier properties.

To date, most evidence for astrocyte contribution to the BBB has been generated in vitro.For example, an unspecified astrocyte-derived factor induces endothelial polarization and pro-duction of the distinct glycocalyx of CNS endothelium (Yamagata et al. 1997). Furthermore,astrocyte-derived Sonic hedgehog (Shh) signaling (a) stimulates the expression of TJ proteinssuch as Claudin-5 and Occludin in endothelial cells (Alvarez et al. 2011) and (b) reduces the ex-pression of LAMs and chemokines, which results in the immune quiescent phenotype of brainendothelial cells (Alvarez et al. 2011).

In vivo evidence for the contribution of astrocytes to BBB formation andmaintenance is sparse.During embryonic development, before astrocytes are present in the brain, Shh is highly expressedby neuroprogenitors, and Shh depletion leads to reduced expression of Claudin-5 in endothelial


Ann

u. R

ev. C

ell D

ev. B

iol.

2019

.35.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

g A

cces

s pr

ovid

ed b

y H

arva

rd U

nive

rsity

on

07/1

5/19

. For

per

sona

l use

onl

y.


cells at embryonic day (E)13.5 (Alvarez et al. 2011). A recent in vivo study investigated anotherpathway of brain endothelial cell communicationwith glial cells during BBBdevelopment (Segarraet al. 2018). This study showed that neuronal Reelin activates Dab1 in endothelial cells, whichcauses endothelial cells to deposit laminin α4 to the ECM.Dab1 conditional knockout mice showbarrier leakage and reduced astrocyte endfeet coverage on brain capillaries. Segarra et al. (2018)concluded that Dab1-activated endothelial cells communicate to glial cells via laminin α4 to sealthe barrier. Overall, this model sheds light on the interaction between endothelial and glial cellsin BBB development. However, further investigation is needed to rule out that endothelial Dab1deletion causes cell-autonomous impairment of the BBB.

Regarding postnatal barrier maintenance by astrocytes, current literature also implies a rolefor Shh signaling. Endothelium-specific deletion of the Shh receptor Smoothened reduces bar-rier integrity but does not affect vessel patterning in the brain (Alvarez et al. 2011). However,although Alvarez et al. (2011) suggested that Shh in the postnatal brain is derived from astrocytes,recent transcriptional profiling studies of different cell types in the CNS showed very low mRNAexpression of Shh in astrocytes (Zeisel et al. 2018, Zhang et al. 2014), challenging the proposedmechanism.

Another contribution of astrocytes to barrier maintenance was demonstrated by Yao et al.(2014), who showed that laminins specifically secreted by astrocytes maintain BBB integrity.Dele-tion of astrocytic laminins leads to decreased Aqp4 expression in astrocytes and to reduced TJ pro-tein levels in endothelial cells. Mechanistically, Yao et al. (2014) suggest that astrocytic lamininsinhibit pericyte differentiation via integrin α2 activation, which results in a leaky barrier.

In summary, there are many indications for a contribution of astrocytes to BBB formation andmaintenance, in addition to the intriguing close proximity of astrocytic endfeet to the vasculature.However, the exact signaling pathways and mechanisms require a more detailed examination.

3.3. Basement Membrane of the Neurovascular Unit

The BM is the ECM that provides structural support for the cells of the NVU and is a hub forintercellular communication and signaling pathways between these cells. Structural proteins thatmake up the ECM of the BM are type IV collagens, fibronectin, laminins, and other glycopro-teins. Collagen IV and fibronectin are secreted by all three cell types of the NVU, and deletionof either of these genes is embryonic lethal (George et al. 1993, Poschl et al. 2004). Laminins,in contrast, have several isoforms, and the appropriate balance of these isoforms is important invessel formation (Thyboll et al. 2002), barrier formation (Menezes et al. 2014), and the regulationof leukocyte infiltration into the brain (Wu et al. 2009).

The BM has two main families of ECM receptors: dystroglycans and integrins. Together, thesereceptors enable cell-ECM interactions and link the ECM to the cytoskeleton.The ligands withinthe ECM bind to these matrix receptors to trigger signaling cascades regulating cell migration,proliferation, and cell survival.These signaling cascades in turn regulate barrier function. Integrinsare ligand-binding membrane proteins that are heterodimers comprising α and β subunits. Theαv subunit is one of the best-studied integrin subunits in the field owing to its role in angiogen-esis. Itgav−/− mice die of cerebral hemorrhages (Bader et al. 1998), and interestingly, conditionalknockouts revealed that deletion of Itgav from neurons and glia, but not from endothelial cellsor pericytes, recapitulated the phenotype (McCarty et al. 2002, 2005). Furthermore, studies havealso shown that αv integrins regulate the brain vasculature through activation of TGF-β sig-naling (reviewed in Munger & Sheppard 2011, Sheppard 2004). There is also growing evidencefor integrin-mediated and ECM-driven regulation of Wnt signaling (Astudillo & Larrain 2014),which in turn modulates barrier properties. For example, deletion of integrin β1 in endothelial


Ann

u. R

ev. C

ell D

ev. B

iol.

2019

.35.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

g A

cces

s pr

ovid

ed b

y H

arva

rd U

nive

rsity

on

07/1

5/19

. For

per

sona

l use

onl

y.


cells impairs VE-cadherin signaling, and such impairment causes disorganization of TJs, resultingin a leaky barrier (Yamamoto et al. 2015).

Thus, the signaling pathways and cellular interactions occurring within the BM of the NVUare coordinated to regulate barrier function. Not surprisingly, BM breakdown is seen in severalCNS disorders and diseases (Thomsen et al. 2017).This degradation is thought to be due tomatrixmetalloproteinases (MMPs), which are upregulated in disease conditions. Specifically, increasedMMP-2 andMMP-9 have consistently been associated with stroke, ischemia, and Alzheimer’s andParkinson’s diseases (Rempe et al. 2016). Although targeting MMPs in disease to impede proteindegradation is an attractive idea from a therapeutic standpoint, studying MMPs has been difficultowing to the lack of suitable reagents to examine their localization or measure their proteolyticactivities in vivo. New tools and further research are needed to understand the functional roles ofthese molecules in BBB regulation.

4. DEVELOPMENT OF THE BLOOD-BRAIN BARRIER

4.1. Barriergenesis

When brain endothelial cells first enter the CNS, they do not inherently display barrier prop-erties. Instead, BBB formation is a gradual process that occurs during embryonic developmentafter exposure to the neural environment. The process begins with vascularization of the brain atapproximately E11.5. Blood vessels from the perineural vascular plexus (PNVP) invade the brainin a stereotypical manner by sprouting angiogenesis (Daneman et al. 2010, Risau 1997). Vascu-larization is initiated by the release of growth factors from the neural tube. These factors attractvessels to invade via binding to specific receptors on the endothelial cell surface. Shortly after vas-cular sprouts invade the neural tube, they begin to connect and form a vessel plexus in the brain(Engelhardt 2003).

Numerous signaling pathways such as the VEGF, Notch, and ephrin signaling pathways playcrucial roles in CNS angiogenesis (Engelhardt & Liebner 2014).However, these pathways are notspecific to CNS vascularization. Perturbations in these signaling pathways lead to severe vasculardefects both in the CNS and in peripheral organs, including the most severe form of a barrierbreakdown: hemorrhage. Consequently, these phenotypes are likely due to angiogenic defectsthroughout all tissues as opposed to CNS-specific angiogenic mechanisms.

An elegant model to study barriergenesis specifically in the CNS is the mouse retina. Theretina is vascularized radially from the central nerve from postnatal day (P)0 to P10 (Chow & Gu2017, Pitulescu et al. 2010, Stahl et al. 2010). Chow & Gu (2017) recently showed by ultrastruc-tural and molecular investigation that invading vessel sprouts already have functional TJs yet haveactive transcytosis and therefore do not have a functional barrier. During vascular outgrowth, theimmature distal vessels in the retina are leaky, while the mature proximal vessels have a sealed bar-rier. Interestingly, expression of Mfsd2a, a molecule that suppresses transcytosis, correlates withfunctional barrier formation (Chow & Gu 2017). This model proposes a gradual sealing of thebarrier, with manifestation of TJs first and inhibition of vesicular transport later (Figure 2).

While the brain is much more complex than the retina and the chronology of events that sealthe barrier has been debated, barriergenesis appears to follow a similar gradual process. Initialsprouts invading into the neural tube from the PNVP display TJs (Risau 1991, Risau et al. 1986)and already show expression of genes associated with barrier function such as Slc2a1, Cldn5, andAbcb1a (Dermietzel et al. 1992, Morita et al. 1999, Qin & Sato 1995). At the same time, murinebrain vessels contain transcytotic vesicles before E15.5 (Ben-Zvi et al. 2014). Moreover, up toE15.5 capillaries express Plvap, a marker associated with transcytosis and endothelial fenestrae


Ann

u. R

ev. C

ell D

ev. B

iol.

2019

.35.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

g A

cces

s pr

ovid

ed b

y H

arva

rd U

nive

rsity

on

07/1

5/19

. For

per

sona

l use

onl

y.


Lumen

Lumen

EC

Immature leaky BBB

Wnt/NdpWnt/Ndp

EC

Mature sealed BBB

Wnt/NdpWnt/NdpMfsd2a Retina P7Brain E14.5

Parenchyma

Parenchyma

Dorsal

Ventral

Distal

ProximalProximal

Tightjunctions

Vesiculartransport

β-Catenin

Tightjunctions

β-Catenin

Lumen

Figure 2

Gradual formation of the blood-brain barrier (BBB) and blood-retina barrier. Depiction of a coronal brain section at E14.5 (left) and aflat-mounted retina at P7 (right) highlighting the spatiotemporal gradient in functional barrier formation in the CNS endothelial cells(ECs). The top inset (outlined in blue) illustrates features of the immature, leaky CNS barriers found in dorsal brain regions and thedistal retina. Canonical Wnt signaling induces tight junction formation and prevents paracellular flow. However, transcellular flow viatranscytosis remains active. The bottom inset (outlined in green) shows features of the mature, sealed blood-CNS barriers of ventralembryonic brain and the proximal vessels of the developing retina. Here, in addition to Wnt signaling, Mfsd2a expression inhibitstranscellular flow through transcytosis.

(Daneman et al. 2010, Hallmann et al. 1995, Stan et al. 2012). Tracer injections have shown thatbrain vessels show leakage of tracers only until E15.5 (Ben-Zvi et al. 2014, Risau et al. 1986). In-terestingly, the functional BBB forms in a ventral-to-dorsal fashion (Ben-Zvi et al. 2014,Danemanet al. 2010), indicating spatiotemporal maturation of the barrier (Figure 2).

To summarize, when blood vessels first enter the CNS, they immediately acquire functionalTJs. This process is mainly controlled by the canonical Wnt signaling pathway, discussed below.In contrast, transcellular transport through endothelial cells via transcytosis is active during earlierstages of development and is then gradually suppressed.Thus, the formation of a functional barriercoincides with the suppression of transcytosis (Figure 2).

Interestingly, while the brain is growing and angiogenesis is still occurring, newly formingvessels immediately display the barrier phenotype. This phenomenon is best demonstrated in theretina.When the intermediate plexus is vascularized between P12 and P17, vessel sprouts invadingfrom the deeper plexus into the intermediate plexus are not leaky, although they are still under-going angiogenesis (Chow & Gu 2017). Thus, newly formed endothelial cells after initial barrierformation inherit barrier properties from their mother cell, and they depend on the local microen-vironment to maintain these barrier properties ( Janzer & Raff 1987, Stewart & Wiley 1981).


Ann

u. R

ev. C

ell D

ev. B

iol.

2019

.35.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

g A

cces

s pr

ovid

ed b

y H

arva

rd U

nive

rsity

on

07/1

5/19

. For

per

sona

l use

onl

y.


4.2. Wnt Signaling

Canonical Wnt signaling is, as currently known, the major pathway specifically regulating brainangiogenesis and barriergenesis, but not peripheral angiogenesis (Daneman et al. 2009, Liebneret al. 2008, Moro et al. 2012, Stenman et al. 2008). During development, neural progenitor cellssecrete Wnt ligands, which act on Frizzled receptors on endothelial cells, activating canonicalWnt signaling (Daneman et al. 2009, Stenman et al. 2008). In this pathway, Wnt ligands bindingto their receptors induce β-catenin stabilization and downstream gene induction via TCF/LEFtranscription factors.

Endothelium-specific knockout mice for the canonical Wnt signaling mediator β-catenin(Ctnnb1) show embryonic lethality associated with severe hemorrhaging specifically in the CNS(Daneman et al. 2009). Early postnatal endothelial cell–specific deletion of Ctnnb1 disrupts bar-rier integrity, which correlates with reduced Claudin-5 expression (Liebner et al. 2008, Zhou et al.2014). Multiple studies have illustrated that the expression of marker genes for a functioning bar-rier such as Slc2a1 and Cldn5 and the absence of Plvap are directly regulated by Wnt signaling(Daneman et al. 2009, Liebner et al. 2008, Zhou et al. 2014).

To date, at least two ligand-receptor signaling complexes have been identified that mediate β-catenin stabilization and thereby CNS vascularization and barriergenesis. These pathways showpartially redundant functions in activating canonical Wnt signaling.

In the first pathway, Wnt7a/b ligands signal through Frizzled receptors, which are in com-plex with their coreceptors Lrp5/6, GPR124, and Reck to induce canonical Wnt signaling(Table 1). Wnt7a and Wnt7b are expressed by astroglia, oligodendrocytes, and neurons in theforebrain and ventral neural tube (Daneman et al. 2009, Wang et al. 2018, Zhang et al. 2014).They act on Frizzled receptors, which are located in complex with their coreceptors on endothe-lial cells (C. Cho et al. 2017, Eubelen et al. 2018, Zhou et al. 2014). Knockout mice for com-ponents of this pathway show CNS angiogenic defects and leaky CNS barriers. However, theseeffects occur in distinct regions of the CNS, namely the cerebral cortex and dorsal spinal cord(C. Cho et al. 2017, Daneman et al. 2009, Stenman et al. 2008, Wang et al. 2012, Zhou et al.2014).

The role of GPR124, the coactivator in this pathway, has been well studied (Anderson et al.2011, Chang et al. 2017, Cullen et al. 2011, Kuhnert et al. 2010, Vanhollebeke et al. 2015, Zhouet al. 2014). Interestingly, while GPR124 plays a major role in BBB development, with knockoutmice displaying severe angiogenic defects and tracer leakage in the forebrain, endothelium-specificdeletion in adult mice does not result in barrier breakdown (Chang et al. 2017). In contrast, post-natal deletion of either Ctnnb1 or Fzd4 (which encodes Frizzled4) results in a compromised BBB(Tran et al. 2016,Wang et al. 2012). However, under challenge, mouse models of ischemic strokeor glioblastoma GPR124 deficiency show rapid barrier breakdown via loss of canonical Wnt sig-naling (Chang et al. 2017).

The second describedWnt pathway in barriergenesis is activated by Norrin (encoded byNdp),a ligand that belongs to the TGFβ family and that binds to Frizzled4 (Smallwood et al. 2007).Norrin is expressed by astroglia and, to some extent, by oligodendrocytes in the CNS (Zhanget al. 2014). Binding of Norrin to Frizzled4, which acts with its coreceptors Lrp5 and Tspan12,leads to downstream β-catenin stabilization ( Junge et al. 2009, Wang et al. 2012, Ye et al. 2010)(Table 1). Knockout mice for Ndp, Fzd4, or Tspan12 show severe angiogenic phenotypes andhave a dysfunctional barrier in the retina, cerebellum, and ventral spinal cord ( Junge et al. 2009,Wang et al. 2012). In the retina, the Wnt inhibitor Apcdd1 modulates the Norrin pathway andbarrier maturation (Mazzoni et al. 2017). Depleting components of both the Norrin andWnt7a/bpathways during development results in severe leakage throughout the brain, including regions


Ann

u. R

ev. C

ell D

ev. B

iol.

2019

.35.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

g A

cces

s pr

ovid

ed b

y H

arva

rd U

nive

rsity

on

07/1

5/19

. For

per

sona

l use

onl

y.


Table 1 Determinants of blood-brain barrier physiology

Cell type Protein (gene) Function Reference(s)Endothelial

cellsLsr (Lsr) Formation of tricellular junctions Ikenouchi et al. 2005, Sohet et al.

2015Claudin-5 (Cldn5) Formation of tight junctions Morita et al. 1999, Nitta et al. 2003Glut1 (Slc2a1) Transport of glucose across the barrier De Vivo et al. 1991,Wang et al.

2006,Winkler et al. 2015LAT1 (Slc7a5) Transport of branched amino acids across the

barrierTarlungeanu et al. 2016

P-glycoprotein(Abcb1)

Export of amyloid β and efflux of drugs from thebrain

Cirrito et al. 2005, Cordon-Cardoet al. 1989, Schinkel et al. 1996

Mfsd2a (Mfsd2a) Suppression of transcytosis Andreone et al. 2017, Ben-Zvi et al.2014

Transferrin receptor(Tfrc)

Receptor for transferrin; undergoes transcytosisupon binding of transferrin

Friden et al. 1991, Jefferies et al.1984, Yu & Watts 2013

PDGF-B (Pdgfb) Recruitment of pericytes Armulik et al. 2010, Hellstrom et al.1999, Lindahl et al. 1997

Smoothened (Smo) Activation by Sonic hedgehog induces Cldn5 andOcln expression and induces immunequiescence

Alvarez et al. 2011

Dab1 (Dab1) Activation by Reelin induces laminin α4localization to ECM and astrocyte endfeetattachment

Segarra et al. 2018

Integrin β1 (Itgb1) Required for VE-cadherin localization toadherens junctions

Yamamoto et al. 2015

Lrp5/6 (Lrp5, Lrp6) Coreceptors of Frizzled receptors; required toinduce tight junction formation via β-cateninstabilization

Chen et al. 2012, Junge et al. 2009,Zhou et al. 2014

GPR124 (Adgra2) Coreceptor of Frizzled receptors; required toinduce tight junction formation via β-cateninstabilization

Anderson et al. 2011, Chang et al.2017, C. Cho et al. 2017

Reck (Reck) Coreceptor of Frizzled receptors; required toinduce tight junction formation via β-cateninstabilization

C. Cho et al. 2017, Vanhollebekeet al. 2015

Frizzled4 (Fzd4) Frizzled receptor mediating Norrin signal toinduce tight junction formation via β-cateninstabilization

Smallwood et al. 2007,Wang et al.2018

Tspan12 (Tspan12) Coreceptor of Frizzled4; required to induce tightjunction formation via β-catenin stabilization

Junge et al. 2009,Wang et al. 2018

Pericytes PDGFRβ (Pdgfrb) Localization of pericytes to endothelial cells Daneman et al. 2010, Hellstromet al. 1999

Foxf2 (Foxf2) Stimulates Pdgfrb expression and TGFβ signaling Reyahi et al. 2015Astrocytes Sonic hedgehog (Shh) Activates Smoothened on endothelial cells and

induces Cldn5 and Ocln expression and immunequiescence

Alvarez et al. 2011

Laminin γ1 (Lamc1) Induces tight junction protein expression inendothelial cells and Aqp4 expression inastrocytes

Yao et al. 2014

(Continued)


Ann

u. R

ev. C

ell D

ev. B

iol.

2019

.35.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

g A

cces

s pr

ovid

ed b

y H

arva

rd U

nive

rsity

on

07/1

5/19

. For

per

sona

l use

onl

y.


Table 1 (Continued)

Cell type Protein (gene) Function Reference(s)Other celltypes

Reelin (Reln) Secreted by neuronal cells; activates Dab1 inendothelial cells

Segarra et al. 2018

Wnt7a and -b(Wnt7a,Wnt7b)

Secreted by Bergmann glia and neuronal cells;activate Frizzled receptors, inducing canonicalβ-catenin signaling

Daneman et al. 2009,Wang et al.2018

Norrin (Ndp) TGFβ family ligand; secreted by neuronal cellsand astroglia; activates Frizzled4

Smallwood et al. 2007; Wang et al.2012, 2018

Sonic hedgehog (Shh) Secreted by neural progenitors and astrocytes;activates Smoothened on endothelial cells

Alvarez et al. 2011

such as the brain stem that are not affected by perturbation of either pathway alone (C. Cho et al.2017, Wang et al. 2018, Zhou et al. 2014).

The two different pathways of canonical Wnt activation are regionally active in both mutu-ally exclusive and redundant patterns. The Wnt7a/b pathway is exclusively active in the fore-brain and ventral spinal cord, while the norrin pathway is exclusive to the cerebellum, retina, anddorsal spinal cord. In the brain stem, both pathways are active (C. Cho et al. 2017, Wang et al.2018, Zhou et al. 2014). This local regulation of Wnt signaling may provide avenues to manipu-late CNS barriers in a spatially selective manner. Future studies are required to explore this ideafurther.

Canonical Wnt signaling is the best-characterized pathway in BBB formation. The expressionof certain transporters and TJ proteins such as Glut1 and Claudin-5 also correlates with canonicalWnt activation (Figure 2). However, the mechanisms of how canonical Wnt activation leads toformation of a functional barrier are not well understood. For example, it is not known whetherWnt signaling also plays a role in the inhibition of transcytosis, although mutants for Wnt com-ponents show increased Plvap expression. Plvap expression commonly correlates with permeablevasculature and has been associated with transcytosis; however, overexpression of Plvap does notincrease brain vessel permeability (Stan et al. 2012).

While we are beginning to understand the contribution of canonical Wnt signaling to BBBdevelopment, there are likely other involved pathways that are locally integrated into the forma-tion of a functional barrier. One task for future research will be to understand how these pathwayscollaborate throughout the CNS and locally in defined brain regions.

5. OUTLOOK

Other mammalian tissues, such as the gut, skin, placenta, testis, and kidney, also contain barriers.These barriers regulate the molecular exchange between neighboring compartments, althoughwith various levels of permeability, depending on the function of the tissue. Because each barrieroperates in a distinct tissue environment, the site of the barrier is formed by different cell typesin each tissue. For example, while endothelial cells form the BBB, epithelial cells form the gutbarrier, and Sertoli cells form the testis barrier. However, these barriers and the BBB also exhibitmany similarities. For example, these barriers utilize commonmechanisms to regulate paracellularand transcellular passage, such as by the formation of TJs and control of the transcytotic pathway.Moreover, the cells that compose each of these barriers are polarized and rely on mechanisms ofapical-basolateral sorting. Furthermore, the interaction of each barrier with the ECM and localcellular microenvironment dynamically regulates the function of each of these organ barriers.


Ann

u. R

ev. C

ell D

ev. B

iol.

2019

.35.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

g A

cces

s pr

ovid

ed b

y H

arva

rd U

nive

rsity

on

07/1

5/19

. For

per

sona

l use

onl

y.


Within the field of BBB research, we envision several new avenues that will unravel previouslyunknown modes of BBB regulation. The advent of new single-cell transcriptomic analyses has al-lowed researchers to begin to identify the various cell types in the brain and their transcriptionalsignatures (Saunders et al. 2018, Vanlandewijck et al. 2018, Zeisel et al. 2018). Our next chal-lenges will be to utilize these data sets to identify the specific molecules and signaling pathwaysthat mediate BBB formation and function across cell types. Additionally, identifying endothelialsubtype–specific markers might help in understanding the spatial distribution of cell subtypes andin dissecting regional differences in barrier induction. The development of genetic tools to targetpericytes, astrocytes, or subtypes of endothelial cells will be critical to advancing our understand-ing of barrier function.

As discussed in this review, the BBB is a highly dynamic structure regulated by multiple celltypes.The latest research has found that BBB properties in fruit flies underlie the circadian rhythm(Zhang et al. 2018). While the structure of the barrier is very different in flies than in mammals(O’Brown et al. 2018), it will be interesting to see whether the capacity of barrier modulationby circadian signals is conserved across species. Although we are beginning to dissect specificroles of different cell types during barrier development, their differential contributions to barriermaintenance through adulthood are unclear. Furthermore, the physiology of the barrier and theroles of pericytes and astrocytes in disease progression are unknown. Although barrier breakdownhas been observed in several pathologies, it is unclear whether breakdown is the initial step ofpathogenesis or a consequence of disease progression.

The discovery of novel BBB regulators and a deeper understanding of the mechanisms medi-ating barrier function will likely provide new targets to pharmacologically modulate the barrier indisease conditions. After this initial identification, reliable and robust in vitro systems are neededto develop high-throughput screening of molecules that can change barrier properties. In recentyears, a fewmodels have been successful in capturingmost, if not all, properties of theNVU.Thesemodels include inducible pluripotent stem cells (Lippmann et al. 2012), self-assembling multicel-lular BBB spheroids (C.-F. Cho et al. 2017), and microfluidic organ chips (Maoz et al. 2018). Itremains to be seen whether we can use these model systems to screen molecules for drug deliveryinto the brain and to study the basic cell biology of the NVU, which can then be investigated invivo.

Finally, the heterogeneity of the BBB across various brain regions is an unexplored area of re-search that needs investigation. The phenotypes of mouse mutants for the Wnt signaling compo-nents described above demonstrate that there is molecular heterogeneity in BBB formation acrossdifferent brain regions. It is presently unknown whether differential development translates intodistinct barrier properties during adulthood. Moreover, there are regions in the brain with per-meable vessels serving nearby neurons to sense components in the bloodstream and to secretecompounds into circulation. Understanding the molecular and cellular basis of a functional bar-rier in different parts of the brain not only will identify new players in BBB function but also willuncover unique themes of barrier regulation. Together, achieving such advances would have thepotential to provide novel ways to manipulate the barrier globally as well as in a region-specificmanner. Over the next few decades, we anticipate all these research areas to reveal new aspectsof barrier regulation that will provide a detailed understanding of the BBB in both health anddisease.

DISCLOSURE STATEMENT

The authors are not aware of any affiliations, memberships, funding, or financial holdings thatmight be perceived as affecting the objectivity of this review.


Ann

u. R

ev. C

ell D

ev. B

iol.

2019

.35.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

g A

cces

s pr

ovid

ed b

y H

arva

rd U

nive

rsity

on

07/1

5/19

. For

per

sona

l use

onl

y.


ACKNOWLEDGMENTS

We thank members of the Gu laboratory for helpful comments on the article. We apologize toour colleagues whose research we could not cite or discuss owing to space limitations. This workwas funded by an EMBO long-term fellowship to U.H.L., by a Mahoney postdoctoral fellowshipto S.A., and by National Institutes of Health grant DP1NS092473 to C.G. The research of C.G.was also supported in part by a Faculty Scholar grant from the Howard Hughes Medical Institute.

LITERATURE CITED

Alvarez JI, Dodelet-Devillers A, Kebir H, Ifergan I, Fabre PJ, et al. 2011. The Hedgehog pathway promotesblood-brain barrier integrity and CNS immune quiescence. Science 334:1727–31

Anderson KD, Pan L, Yang XM, Hughes VC, Walls JR, et al. 2011. Angiogenic sprouting into neural tissuerequires Gpr124, an orphan G protein-coupled receptor. PNAS 108:2807–12

Andreone BJ, Chow BW, Tata A, Lacoste B, Ben-Zvi A, et al. 2017. Blood-brain barrier permeability is regu-lated by lipid transport-dependent suppression of caveolae-mediated transcytosis.Neuron 94:581–94.e5

Armulik A, Genove G, Mae M, Nisancioglu MH,Wallgard E, et al. 2010. Pericytes regulate the blood–brainbarrier.Nature 468:557–61

Armulik A,MaeM,Betsholtz C. 2011. Pericytes and the blood–brain barrier: recent advances and implicationsfor the delivery of CNS therapy. Ther. Deliv. 2:419–22

Astudillo P, Larrain J. 2014.Wnt signaling and cell-matrix adhesion. Curr. Mol. Med. 14:209–20Bader BL, Rayburn H, Crowley D, Hynes RO. 1998. Extensive vasculogenesis, angiogenesis, and organogen-

esis precede lethality in mice lacking all αv integrins. Cell 95:507–19Banks WA. 2016. From blood–brain barrier to blood–brain interface: new opportunities for CNS drug deliv-

ery.Nat. Rev. Drug Discov. 15:275–92Beaulieu E, Demeule M, Ghitescu L, Beliveau R. 1997. P-glycoprotein is strongly expressed in the luminal

membranes of the endothelium of blood vessels in the brain. Biochem. J. 326(Part 2):539–44Bell RD,Winkler EA, Sagare AP, Singh I, LaRue B, et al. 2010. Pericytes control key neurovascular functions

and neuronal phenotype in the adult brain and during brain aging.Neuron 68:409–27Ben-Zvi A, Lacoste B, Kur E, Andreone BJ, Mayshar Y, et al. 2014. Mfsd2a is critical for the formation and

function of the blood–brain barrier.Nature 509:507–11Bien-Ly N, Yu YJ, Bumbaca D, Elstrott J, Boswell CA, et al. 2014. Transferrin receptor (TfR) trafficking

determines brain uptake of TfR antibody affinity variants. J. Exp. Med. 211:233–44Birbrair A, Zhang T,Wang ZM,Messi ML,Mintz A, Delbono O. 2015. Pericytes at the intersection between

tissue regeneration and pathology. Clin. Sci. 128:81–93Brightman MW, Reese TS. 1969. Junctions between intimately apposed cell membranes in the vertebrate

brain. J. Cell Biol. 40:648–77Broadwell RD, Salcman M. 1981. Expanding the definition of the blood-brain barrier to protein. PNAS

78:7820–24Campos-Bedolla P,Walter FR, Veszelka S, Deli MA. 2014. Role of the blood–brain barrier in the nutrition of

the central nervous system. Arch. Med. Res. 45:610–38Cannella B, Cross AH, Raine CS. 1990. Upregulation and coexpression of adhesion molecules correlate with

relapsing autoimmune demyelination in the central nervous system. J. Exp. Med. 172:1521–24Castejon OJ, Castellano A, Arismendi GJ, Medina Z. 2005. The inflammatory reaction in human traumatic

oedematous cerebral cortex. J. Submicrosc. Cytol. Pathol. 37:43–52Chang J, Mancuso MR, Maier C, Liang X, Yuki K, et al. 2017. Gpr124 is essential for blood–brain barrier

integrity in central nervous system disease.Nat. Med. 23:450–60Chen J, Stahl A, Krah NM, Seaward MR, Joyal JS, et al. 2012. Retinal expression of Wnt-pathway mediated

genes in low-density lipoprotein receptor-related protein 5 (Lrp5) knockout mice. PLOS ONE 7:e30203ChoC,Smallwood PM,Nathans J. 2017.Reck andGpr124 are essential receptor cofactors forWnt7a/Wnt7b-

specific signaling in mammalian CNS angiogenesis and blood-brain barrier regulation.Neuron 95:1056–73.e5


Ann

u. R

ev. C

ell D

ev. B

iol.

2019

.35.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

g A

cces

s pr

ovid

ed b

y H

arva

rd U

nive

rsity

on

07/1

5/19

. For

per

sona

l use

onl

y.


Cho C-F,Wolfe JM, Fadzen CM, Calligaris D, Hornburg K, et al. 2017. Blood-brain-barrier spheroids as anin vitro screening platform for brain-penetrating agents.Nat. Commun. 8:15623

Chow BW, Gu C. 2017. Gradual suppression of transcytosis governs functional blood-retinal barrier forma-tion.Neuron 93:1325–33.e3

Cirrito JR, Deane R, Fagan AM, Spinner ML, Parsadanian M, et al. 2005. P-glycoprotein deficiency at theblood-brain barrier increases amyloid-βdeposition in anAlzheimer diseasemousemodel.J.Clin. Investig.115:3285–90

Cordon-Cardo C,O’Brien JP, Casals D, Rittman-Grauer L, Biedler JL, et al. 1989.Multidrug-resistance gene(P-glycoprotein) is expressed by endothelial cells at blood-brain barrier sites. PNAS 86:695–98

Cullen M, Elzarrad MK, Seaman S, Zudaire E, Stevens J, et al. 2011. GPR124, an orphan G protein-coupledreceptor, is required forCNS-specific vascularization and establishment of the blood–brain barrier.PNAS108:5759–64

Damisah EC, Hill RA, Tong L, Murray KN, Grutzendler J. 2017. A fluoro-Nissl dye identifies pericytes asdistinct vascular mural cells during in vivo brain imaging.Nat. Neurosci. 20:1023–32

Daneman R, Agalliu D, Zhou L, Kuhnert F, Kuo CJ, Barres BA. 2009. Wnt/β-catenin signaling is requiredfor CNS, but not non-CNS, angiogenesis. PNAS 106:641–46

Daneman R, Zhou L, Kebede AA, Barres BA. 2010. Pericytes are required for blood–brain barrier integrityduring embryogenesis.Nature 468:562–66

De Vivo DC, Trifiletti RR, Jacobson RI, Ronen GM, Behmand RA, Harik SI. 1991. Defective glucose trans-port across the blood-brain barrier as a cause of persistent hypoglycorrhachia, seizures, and developmen-tal delay.N. Engl. J. Med. 325:703–9

Deane R, Wu Z, Sagare A, Davis J, Du Yan S, et al. 2004. LRP/amyloid β-peptide interaction mediates dif-ferential brain efflux of Aβ isoforms.Neuron 43:333–44

Dermietzel R, Krause D, Kremer M,Wang C, Stevenson B. 1992. Pattern of glucose transporter (Glut 1) ex-pression in embryonic brains is related tomaturation of blood-brain barrier tightness.Dev.Dyn.193:152–63

Dore-Duffy P. 2008. Pericytes: pluripotent cells of the blood brain barrier. Curr. Pharm. Des. 14:1581–93Duan L, Zhang XD,MiaoWY, Sun YJ, Xiong G, et al. 2018. PDGFRβcells rapidly relay inflammatory signal

from the circulatory system to neurons via chemokine CCL2.Neuron 100:183–200.e8Eberth CJ. 1871.Handbuch der Lehre von der Geweben des Menschen und der Tiere. Leipzig: EngelmannEdqvist PH, Niklasson M, Vidal-Sanz M, Hallbook F, Forsberg-Nilsson K. 2012. Platelet-derived growth

factor over-expression in retinal progenitors results in abnormal retinal vessel formation. PLOS ONE7:e42488

Engelhardt B. 2003. Development of the blood-brain barrier. Cell Tissue Res. 314:119–29Engelhardt B, Liebner S. 2014. Novel insights into the development and maintenance of the blood–brain

barrier. Cell Tissue Res. 355:687–99Engelhardt B, Wolburg H. 2004. Mini-review: Transendothelial migration of leukocytes: through the front

door or around the side of the house? Eur. J. Immunol. 34:2955–63Eubelen M, Bostaille N, Cabochette P, Gauquier A, Tebabi P, et al. 2018. A molecular mechanism for Wnt

ligand-specific signaling. Science 361:eaat1178Farquhar MG, Palade GE. 1963. Junctional complexes in various epithelia. J. Cell Biol. 17:375–412Frank RN, Dutta S, Mancini MA. 1987. Pericyte coverage is greater in the retinal than in the cerebral capil-

laries of the rat. Investig. Ophthalmol. Vis. Sci. 28:1086–91Frank RN, Turczyn TJ, Das A. 1990. Pericyte coverage of retinal and cerebral capillaries. Investig. Ophthalmol.

Vis. Sci. 31:999–1007Friden PM,Walus LR, Musso GF, Taylor MA, Malfroy B, Starzyk RM. 1991. Anti-transferrin receptor anti-

body and antibody-drug conjugates cross the blood–brain barrier. PNAS 88:4771–75Furuse M, Fujita K, Hiiragi T, Fujimoto K, Tsukita S. 1998. Claudin-1 and -2: novel integral membrane

proteins localizing at tight junctions with no sequence similarity to occludin. J. Cell Biol. 141:1539–50

Furuse M, Hirase T, Itoh M, Nagafuchi A, Yonemura S, et al. 1993. Occludin: a novel integral membraneprotein localizing at tight junctions. J. Cell Biol. 123:1777–88


Ann

u. R

ev. C

ell D

ev. B

iol.

2019

.35.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

g A

cces

s pr

ovid

ed b

y H

arva

rd U

nive

rsity

on

07/1

5/19

. For

per

sona

l use

onl

y.


Gaengel K,GenoveG,Armulik A,Betsholtz C.2009.Endothelial-mural cell signaling in vascular developmentand angiogenesis. Arterioscler. Thromb. Vasc. Biol. 29:630–38

George EL, Georges-Labouesse EN, Patel-King RS, Rayburn H, Hynes RO. 1993. Defects in mesoderm,neural tube and vascular development in mouse embryos lacking fibronectin.Development 119:1079–91

Haley MJ, Lawrence CB. 2017. The blood–brain barrier after stroke: structural studies and the role of tran-scytotic vesicles. J. Cereb. Blood Flow Metab. 37:456–70

Hallmann R, Mayer DN, Berg EL, Broermann R, Butcher EC. 1995. Novel mouse endothelial cell surfacemarker is suppressed during differentiation of the blood brain barrier.Dev. Dyn. 202:325–32

Hansen CG, Nichols BJ. 2009. Molecular mechanisms of clathrin-independent endocytosis. J. Cell Sci.122:1713–21

Hauser SL, Bhan AK, Gilles FH, Hoban CJ, Reinherz EL, et al. 1983. Immunohistochemical staining ofhuman brain with monoclonal antibodies that identify lymphocytes, monocytes, and the Ia antigen.J. Neuroimmunol. 5:197–205

Hediger MA, Clemencon B, Burrier RE, Bruford EA. 2013. The ABCs of membrane transporters in healthand disease (SLC series): introduction.Mol. Aspects Med. 34:95–107

Hellstrom M, Kalen M, Lindahl P, Abramsson A, Betsholtz C. 1999. Role of PDGF-B and PDGFR-β inrecruitment of vascular smooth muscle cells and pericytes during embryonic blood vessel formation inthe mouse.Development 126:3047–55

Hickey WF, Hsu BL, Kimura H. 1991. T-lymphocyte entry into the central nervous system. J. Neurosci. Res.28:254–60

Iadecola C. 2004. Neurovascular regulation in the normal brain and in Alzheimer’s disease.Nat. Rev. Neurosci.5:347–60

Iadecola C. 2017. The neurovascular unit coming of age: a journey through neurovascular coupling in healthand disease.Neuron 96:17–42

Ikenouchi J, Furuse M, Furuse K, Sasaki H, Tsukita S, Tsukita S. 2005. Tricellulin constitutes a novel barrierat tricellular contacts of epithelial cells. J. Cell Biol. 171:939–45

Ikenouchi J, Sasaki H, Tsukita S, Furuse M, Tsukita S. 2008. Loss of occludin affects tricellular localization oftricellulin.Mol. Biol. Cell 19:4687–93

Janzer RC, Raff MC. 1987. Astrocytes induce blood–brain barrier properties in endothelial cells. Nature325:253–57

Jefferies WA, Brandon MR, Hunt SV, Williams AF, Gatter KC, Mason DY. 1984. Transferrin receptor onendothelium of brain capillaries.Nature 312:162–63

Jeynes B, Provias J. 2011. An investigation into the role of P-glycoprotein in Alzheimer’s disease lesion patho-genesis.Neurosci. Lett. 487:389–93

Junge HJ, Yang S, Burton JB, Paes K, Shu X, et al. 2009. TSPAN12 regulates retinal vascular development bypromoting Norrin- but not Wnt-induced FZD4/β-catenin signaling. Cell 139:299–311

Kanekiyo T, Liu CC, Shinohara M, Li J, Bu G. 2012. LRP1 in brain vascular smooth muscle cells mediateslocal clearance of Alzheimer’s amyloid-β. J. Neurosci. 32:16458–65

Knowland D, Arac A, Sekiguchi KJ, Hsu M, Lutz SE, et al. 2014. Stepwise recruitment of transcellular andparacellular pathways underlies blood-brain barrier breakdown in stroke.Neuron 82:603–17

Kolarova H, Ambruzova B, Svihalkova Sindlerova L, Klinke A, Kubala L. 2014. Modulation of endothelialglycocalyx structure under inflammatory conditions.Mediators Inflamm. 2014:694312

Kuhnert F, Mancuso MR, Shamloo A, Wang HT, Choksi V, et al. 2010. Essential regulation of CNS angio-genesis by the orphan G protein-coupled receptor GPR124. Science 330:985–89

Kutuzov N, Flyvbjerg H, Lauritzen M. 2018. Contributions of the glycocalyx, endothelium, and extravascularcompartment to the blood–brain barrier. PNAS 115:E9429–38

Lam FC, Liu R, Lu P, Shapiro AB, Renoir JM, et al. 2001. β-Amyloid efflux mediated by p-glycoprotein.J. Neurochem. 76:1121–28

Lampugnani MG, Resnati M, Raiteri M, Pigott R, Pisacane A, et al. 1992. A novel endothelial-specific mem-brane protein is a marker of cell–cell contacts. J. Cell Biol. 118:1511–22

Leveen P, Pekny M, Gebre-Medhin S, Swolin B, Larsson E, Betsholtz C. 1994. Mice deficient for PDGF Bshow renal, cardiovascular, and hematological abnormalities.Genes Dev. 8:1875–87


Ann

u. R

ev. C

ell D

ev. B

iol.

2019

.35.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

g A

cces

s pr

ovid

ed b

y H

arva

rd U

nive

rsity

on

07/1

5/19

. For

per

sona

l use

onl

y.


Liebner S, Corada M, Bangsow T, Babbage J, Taddei A, et al. 2008. Wnt/β-catenin signaling controls devel-opment of the blood–brain barrier. J. Cell Biol. 183:409–17

Lindahl P, Johansson BR,Leveen P,Betsholtz C. 1997. Pericyte loss andmicroaneurysm formation in PDGF-B-deficient mice. Science 277:242–45

Lippmann ES, Azarin SM, Kay JE, Nessler RA, Wilson HK, et al. 2012. Derivation of blood-brain barrierendothelial cells from human pluripotent stem cells.Nat. Biotechnol. 30:783–91

Maoz BM, Herland A, FitzGerald EA, Grevesse T, Vidoudez C, et al. 2018. A linked organ-on-chip modelof the human neurovascular unit reveals the metabolic coupling of endothelial and neuronal cells. Nat.Biotechnol. 36:865–74

Martin-Padura I, Lostaglio S, Schneemann M, Williams L, Romano M, et al. 1998. Junctional adhesionmolecule, a novel member of the immunoglobulin superfamily that distributes at intercellular junctionsand modulates monocyte transmigration. J. Cell Biol. 142:117–27

MazloM,Gasz B, Szigeti A, Zsombok A,Gallyas F. 2004.Debris of “dark” (compacted) neurones are removedfrom an otherwise undamaged environment mainly by astrocytes via blood vessels. J. Neurocytol. 33:557–67

Mazzoni J, Smith JR, Shahriar S, Cutforth T,Ceja B, Agalliu D. 2017. TheWnt inhibitor Apcdd1 coordinatesvascular remodeling and barrier maturation of retinal blood vessels.Neuron 96:1055–69.e6

McCarty JH, Lacy-Hulbert A, Charest A, Bronson RT, Crowley D, et al. 2005. Selective ablation of αv in-tegrins in the central nervous system leads to cerebral hemorrhage, seizures, axonal degeneration andpremature death.Development 132:165–76

McCarty JH, Monahan-Earley RA, Brown LF, Keller M, Gerhardt H, et al. 2002. Defective associationsbetween blood vessels and brain parenchyma lead to cerebral hemorrhage in mice lacking αv integrins.Mol. Cell. Biol. 22:7667–77

Menard C, PfauML,Hodes GE,Kana V,Wang VX, et al. 2017. Social stress induces neurovascular pathologypromoting depression.Nat. Neurosci. 20:1752–60

Menezes MJ, McClenahan FK, Leiton CV, Aranmolate A, Shan X, Colognato H. 2014. The extracellularmatrix protein laminin α2 regulates the maturation and function of the blood–brain barrier. J. Neurosci.34:15260–80

Morita K, Sasaki H, Furuse M, Tsukita S. 1999. Endothelial claudin: Claudin-5/TMVCF constitutes tightjunction strands in endothelial cells. J. Cell Biol. 147:185–94

Moro E, Ozhan-Kizil G,Mongera A, Beis D,Wierzbicki C, et al. 2012. In vivoWnt signaling tracing througha transgenic biosensor fish reveals novel activity domains.Dev. Biol. 366:327–40

Munger JS, Sheppard D. 2011. Cross talk among TGF-β signaling pathways, integrins, and the extracellularmatrix. Cold Spring Harb. Perspect. Biol. 3:a005017

Nguyen LN,MaD, Shui G,Wong P,Cazenave-Gassiot A, et al. 2014.Mfsd2a is a transporter for the essentialomega-3 fatty acid docosahexaenoic acid.Nature 509:503–6

Niewoehner J, Bohrmann B, Collin L, Urich E, Sade H, et al. 2014. Increased brain penetration and potencyof a therapeutic antibody using a monovalent molecular shuttle.Neuron 81:49–60

Nitta T, Hata M, Gotoh S, Seo Y, Sasaki H, et al. 2003. Size-selective loosening of the blood-brain barrier inclaudin-5–deficient mice. J. Cell Biol. 161:653–60

O’Brown NM, Pfau SJ, Gu C. 2018. Bridging barriers: a comparative look at the blood–brain barrier acrossorganisms.Genes Dev. 32:466–78

O’Kane RL,Martinez-Lopez I, DeJoseph MR, Vina JR,Hawkins RA. 1999.Na+-dependent glutamate trans-porters (EAAT1, EAAT2, and EAAT3) of the blood-brain barrier: a mechanism for glutamate removal.J. Biol. Chem. 274:31891–95

Oldendorf WH, Cornford ME, Brown WJ. 1977. The large apparent work capability of the blood-brainbarrier: a study of the mitochondrial content of capillary endothelial cells in brain and other tissues ofthe rat. Ann. Neurol. 1:409–17

Pardridge WM. 2012. Drug transport across the blood–brain barrier. J. Cereb. Blood Flow Metab. 32:1959–72

Park DY, Lee J, Kim J, Kim K, Hong S, et al. 2017. Plastic roles of pericytes in the blood–retinal barrier.Nat.Commun. 8:15296


Ann

u. R

ev. C

ell D

ev. B

iol.

2019

.35.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

g A

cces

s pr

ovid

ed b

y H

arva

rd U

nive

rsity

on

07/1

5/19

. For

per

sona

l use

onl

y.


Pitulescu ME, Schmidt I, Benedito R, Adams RH. 2010. Inducible gene targeting in the neonatal vasculatureand analysis of retinal angiogenesis in mice.Nat. Protoc. 5:1518–34

Poschl E,Schlotzer-SchrehardtU,Brachvogel B,SaitoK,Ninomiya Y,MayerU.2004.Collagen IV is essentialfor basement membrane stability but dispensable for initiation of its assembly during early development.Development 131:1619–28

Qin Y, Sato TN. 1995. Mouse multidrug resistance 1a/3 gene is the earliest known endothelial cell differen-tiation marker during blood-brain barrier development.Dev. Dyn. 202:172–80

Raine CS, Lee SC, Scheinberg LC, Duijvestin AM, Cross AH. 1990. Adhesion molecules on endothelial cellsin the central nervous system: an emerging area in the neuroimmunology of multiple sclerosis. Clin.Immunol. Immunopathol. 57:173–87

Ransohoff RM, Engelhardt B. 2012. The anatomical and cellular basis of immune surveillance in the centralnervous system.Nat. Rev. Immunol. 12:623–35

Reese TS, Karnovsky MJ. 1967. Fine structural localization of a blood-brain barrier to exogenous peroxidase.J. Cell Biol. 34:207–17

Rempe RG, Hartz AMS, Bauer B. 2016. Matrix metalloproteinases in the brain and blood–brain barrier: ver-satile breakers and makers. J. Cereb. Blood Flow Metab. 36:1481–507

Reyahi A, Nik AM, Ghiami M, Gritli-Linde A, Ponten F, et al. 2015. Foxf2 is required for brain pericytedifferentiation and development and maintenance of the blood-brain barrier.Dev. Cell 34:19–32

RisauW. 1991. Induction of blood-brain barrier endothelial cell differentiation.Ann. N. Y. Acad. Sci. 633:405–19

Risau W. 1997. Mechanisms of angiogenesis.Nature 386:671–74Risau W,Hallmann R, Albrecht U. 1986. Differentiation-dependent expression of proteins in brain endothe-

lium during development of the blood-brain barrier.Dev. Biol. 117:537–45Rossler K, Neuchrist C, Kitz K, Scheiner O, Kraft D, Lassmann H. 1992. Expression of leucocyte adhesion

molecules at the human blood-brain barrier (BBB). J. Neurosci. Res. 31:365–74Rouget C. 1873. Mémoire sur le développement, la structure et les proprietés physiologiques des capillaires

sanguins et lymphatiques. Arch. Physiol. Norm. Path. 5:603–63Sadeghian H, Lacoste B, Qin T, Toussay X, Rosa R, et al. 2018. Spreading depolarizations trigger caveolin-1-

dependent endothelial transcytosis. Ann. Neurol. 84:409–23Sagare AP, Bell RD, Zhao Z,Ma Q,Winkler EA, et al. 2013. Pericyte loss influences Alzheimer-like neurode-

generation in mice.Nat. Commun. 4:2932Saitou M, Fujimoto K, Doi Y, Itoh M, Fujimoto T, et al. 1998. Occludin-deficient embryonic stem cells can

differentiate into polarized epithelial cells bearing tight junctions. J. Cell Biol. 141:397–408Saitou M, Furuse M, Sasaki H, Schulzke JD, Fromm M, et al. 2000. Complex phenotype of mice lacking

occludin, a component of tight junction strands.Mol. Biol. Cell 11:4131–42Saunders A,Macosko EZ,Wysoker A, Goldman M, Krienen FM, et al. 2018.Molecular diversity and special-

izations among the cells of the adult mouse brain. Cell 174:1015–30.e16Schinkel AH,Wagenaar E, Mol CA, van Deemter L. 1996. P-glycoprotein in the blood-brain barrier of mice

influences the brain penetration and pharmacological activity of many drugs. J. Clin. Investig. 97:2517–24Schulze C,Firth JA. 1993. Immunohistochemical localization of adherens junction components in blood-brain

barrier microvessels of the rat. J. Cell Sci. 104(Part 3):773–82Segarra M, Aburto MR, Cop F, Llaó-Cid C, Härtl R, et al. 2018. Endothelial Dab1 signaling orchestrates

neuro-glia-vessel communication in the central nervous system. Science 361:eaao2861Seo MS, Okamoto N, Vinores MA, Vinores SA, Hackett SF, et al. 2000. Photoreceptor-specific expression of

platelet-derived growth factor-B results in traction retinal detachment. Am. J. Pathol. 157:995–1005Sheppard D. 2004. Roles of αv integrins in vascular biology and pulmonary pathology. Curr. Opin. Cell Biol.

16:552–57ShibataM,Yamada S,Kumar SR,CaleroM,Bading J, et al. 2000.Clearance of Alzheimer’s amyloid-β1–40 pep-

tide from brain by LDL receptor-related protein-1 at the blood-brain barrier. J. Clin. Investig. 106:1489–99

Smallwood PM,Williams J, XuQ,Leahy DJ,Nathans J. 2007.Mutational analysis of Norrin-Frizzled4 recog-nition. J. Biol. Chem. 282:4057–68


Ann

u. R

ev. C

ell D

ev. B

iol.

2019

.35.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

g A

cces

s pr

ovid

ed b

y H

arva

rd U

nive

rsity

on

07/1

5/19

. For

per

sona

l use

onl

y.


Sohet F, Lin C, Munji RN, Lee SY, Ruderisch N, et al. 2015. LSR/angulin-1 is a tricellular tight junctionprotein involved in blood–brain barrier formation. J. Cell Biol. 208:703–11

Soriano P. 1994. Abnormal kidney development and hematological disorders in PDGF β-receptor mutantmice.Genes Dev. 8:1888–96

Stahl A,Connor KM, Sapieha P,Chen J,Dennison RJ, et al. 2010.The mouse retina as an angiogenesis model.Investig. Ophthalmol. Vis. Sci. 51:2813–26

Stan RV, Tse D, Deharvengt SJ, Smits NC, Xu Y, et al. 2012. The diaphragms of fenestrated endothelia—gatekeepers of vascular permeability and blood composition.Dev. Cell 23:1203–18

Stenman JM,Rajagopal J,Carroll TJ, IshibashiM,McMahon J,McMahon AP. 2008.CanonicalWnt signalingregulates organ-specific assembly and differentiation of CNS vasculature. Science 322:1247–50

Stewart PA,Wiley MJ. 1981. Developing nervous tissue induces formation of blood-brain barrier characteris-tics in invading endothelial cells: a study using quail-chick transplantation chimeras.Dev. Biol. 84:183–92

Sweeney MD, Sagare AP, Zlokovic BV. 2018. Blood–brain barrier breakdown in Alzheimer disease and otherneurodegenerative disorders.Nat. Rev. Neurol. 14:133–50

Tarlungeanu DC,Deliu E, Dotter CP, Kara M, Janiesch PC, et al. 2016. Impaired amino acid transport at theblood brain barrier is a cause of autism spectrum disorder. Cell 167:1481–94.e18

Thomsen MS, Routhe LJ, Moos T. 2017. The vascular basement membrane in the healthy and pathologicalbrain. J. Cereb. Blood Flow Metab. 37:3300–17

Thyboll J, Kortesmaa J, Cao R, Soininen R, Wang L, et al. 2002. Deletion of the laminin α4 chain leads toimpaired microvessel maturation.Mol. Cell. Biol. 22:1194–202

TranKA,ZhangX,PredescuD,HuangX,MachadoRF, et al. 2016.Endothelialβ-catenin signaling is requiredfor maintaining adult blood–brain barrier integrity and central nervous system homeostasis. Circulation133:177–86

Vanhollebeke B, Stone OA, Bostaille N, Cho C, Zhou Y, et al. 2015. Tip cell-specific requirement for an atyp-ical Gpr124- and Reck-dependent Wnt/β-catenin pathway during brain angiogenesis. eLife 4:e06489

Vanlandewijck M, He L, Mae MA, Andrae J, Ando K, et al. 2018. A molecular atlas of cell types and zonationin the brain vasculature.Nature 554:475–80

Wang D, Pascual JM, Yang H, Engelstad K,Mao X, et al. 2006. A mouse model for Glut-1 haploinsufficiency.Hum. Mol. Genet. 15:1169–79

Wang Y,Cho C,Williams J, Smallwood PM,Zhang C, et al. 2018. Interplay of the Norrin andWnt7a/Wnt7bsignaling systems in blood–brain barrier and blood–retina barrier development and maintenance. PNAS115:E11827–36

Wang Y, Rattner A, Zhou Y, Williams J, Smallwood PM, Nathans J. 2012. Norrin/Frizzled4 signaling inretinal vascular development and blood brain barrier plasticity. Cell 151:1332–44

Wijesuriya HC, Bullock JY, Faull RL, Hladky SB, Barrand MA. 2010. ABC efflux transporters in brain vascu-lature of Alzheimer’s subjects. Brain Res. 1358:228–38

Winkler EA,Nishida Y, Sagare AP, Rege SV, Bell RD, et al. 2015. GLUT1 reductions exacerbate Alzheimer’sdisease vasculo-neuronal dysfunction and degeneration.Nat. Neurosci. 18:521–30

Wu C, Ivars F, Anderson P, Hallmann R, Vestweber D, et al. 2009. Endothelial basement membrane lamininα5 selectively inhibits T lymphocyte extravasation into the brain.Nat. Med. 15:519–27

Yamagata K, Tagami M, Nara Y, Mitani M, Kubota A, et al. 1997. Astrocyte-conditioned medium inducesblood-brain barrier properties in endothelial cells. Clin. Exp. Pharmacol. Physiol. 24:710–13

Yamamoto H, Ehling M, Kato K, Kanai K, van Lessen M, et al. 2015. Integrin β1 controls VE-cadherinlocalization and blood vessel stability.Nat. Commun. 6:6429

Yang Y, Higashimori H, Morel L. 2013. Developmental maturation of astrocytes and pathogenesis of neu-rodevelopmental disorders. J. Neurodev. Disord. 5:22

Yao Y, Chen ZL, Norris EH, Strickland S. 2014. Astrocytic laminin regulates pericyte differentiation andmaintains blood brain barrier integrity.Nat. Commun. 5:3413

Ye X,Wang Y, Nathans J. 2010. The Norrin/Frizzled4 signaling pathway in retinal vascular development anddisease. Trends. Mol. Med. 16:417–25

Yu YJ, Watts RJ. 2013. Developing therapeutic antibodies for neurodegenerative disease. Neurotherapeutics10:459–72


Ann

u. R

ev. C

ell D

ev. B

iol.

2019

.35.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

g A

cces

s pr

ovid

ed b

y H

arva

rd U

nive

rsity

on

07/1

5/19

. For

per

sona

l use

onl

y.


Zeisel A, Hochgerner H, Lonnerberg P, Johnsson A, Memic F, et al. 2018. Molecular architecture of themouse nervous system. Cell 174:999–1014.e22

Zhang SL, Yue Z, Arnold DM, Artiushin G, Sehgal A. 2018. A circadian clock in the blood-brain barrierregulates xenobiotic efflux. Cell 173:130–39.e10

Zhang Y, Chen K, Sloan SA, Bennett ML, Scholze AR, et al. 2014. An RNA-sequencing transcriptome andsplicing database of glia, neurons, and vascular cells of the cerebral cortex. J. Neurosci. 34:11929–47

Zhao Z, Nelson AR, Betsholtz C, Zlokovic BV. 2015. Establishment and dysfunction of the blood-brain bar-rier. Cell 163:1064–78

Zhou Y, Wang Y, Tischfield M, Williams J, Smallwood PM, et al. 2014. Canonical WNT signaling compo-nents in vascular development and barrier formation. J. Clin. Investig. 124:3825–46


Ann

u. R

ev. C

ell D

ev. B

iol.

2019

.35.

Dow

nloa

ded

from

ww

w.a

nnua

lrev

iew

s.or

g A

cces

s pr

ovid

ed b

y H

arva

rd U

nive

rsity

on

07/1

5/19

. For

per

sona

l use

onl

y.

annualreviewofcellanddevelopmentalbiology … · 2019. 7. 15. · cb35ch12_gu arjats.cls july4,2019...

Documents